Neuropathic pain is a prevalent disorder that accompanies a wide spectrum of diseases including cancer, diabetes, traumatic injuries and AIDS. It is a significant clinical problem because the pain is severe and known analgesics have limited clinical efficacy. One of the important discoveries in pain research in the last few years has been the role of ATP activated P2X4 receptors (P2X4Rs) and microglia-neuron interactions in neuropathic pain. During neuropathic pain P2X4R expression is upregulated in microglia located in the dorsal horn of the pain pathway, and reducing P2X4R function alleviates symptoms of neuropathic pain in rodent models of the disorder. However, many fundamental aspects remain unexplored and there is only a rudimentary understanding of microglial P2X4R properties or the mechanisms involved in their upregulation in neuropathic pain. Progress has been hindered because it is not possible to identify and record from P2X4R expressing cells in tissue slices. A method for identifying P2X4R expressing cells in slices and in vivo would enable the rigorous testing of mechanistic hypotheses regarding microglial P2X4Rs in the healthy CNS and during disease processes. Here we seek to generate and thoroughly characterize an optical reporter mouse using the red fluorescent protein tdTomato. The availability of this mouse will allow researchers to directly visualize and record from P2X4R expressing cells within tissue slices and thus explore the cellular mechanisms that determine P2X4R upregulation and thus contribute significantly to the understanding of mechanisms and plasticity changes in the pain pathway during neuropathic pain. Within the scope of two aims we will generate and fully characterize the reporter mouse and exploit it to measure P2X4 responses using fluorescence guided patch-clamp recordings from microglia and neurons. In Aim 1 we will generate P2X4R reporter mice expressing tdTomato fluorescent proteins as well as establish and characterize different founder lines of transgenic mice to map the location and identity of P2X4R expressing cells. In Aim 2 we will study P2X4R expressing cells using patch-clamp electrophysiology in tissue slices from reporter mice. We will directly test the hypothesis that P2X4R upregulation is specific to activated microglia in the dorsal horn pain pathway. Overall, our approach will provide novel, well characterized optical reporter mice that will allow us and other researchers to identify and record from P2X4R expressing cells within intact tissue structures such as slices of CNS. These new reporter mice will be valuable general tools for the P2X, microglia and pain research communities.